Advances in DNA technology and sequencing, specifically the sequencing of whole genomes including the human genome, have resulted in a significantly increased need to detect and/or quantify specific nucleic acid sequences. Applications include the fields of in vitro diagnostics, including clinical diagnostics, research in the fields of molecular biology, high throughput drug screening, veterinary diagnostics, agricultural-genetics testing, environmental testing, food testing, industrial process monitoring and insurance testing. In vitro diagnostics and clinical diagnostics is related to the analysis of nucleic acid samples drawn from the body to detect the existence of a disease or condition, its stage of development and/or severity, and the patient's response to treatment. In high throughput drug screening and development nucleic acids are used similarly to other agents, such as, antigens, antibodies, receptors, etc, to analyze the response of biological systems upon exposure to libraries of compounds in a high sample number setting to identify drug leads. Veterinary diagnostics and agricultural genetics testing involve samples from a non-human animal or a plant species similar to in vitro diagnostics and to provide means of quality control for agricultural genetic products and processes. In environmental testing, organisms and their toxins that indicate the pollution of an environmental medium, e.g. soil, water, air, etc., are analyzed. Food testing includes the quantitation of organisms, e.g. bacteria, fungi, etc., as a means of quality control. In industrial process monitoring, nucleic acids are detected and/or quantified to indicate proper control of a production process and/or to generate a signal if such processes are out of control. In insurance testing organisms and/or their toxins are identified in screening tests to determine the risk category of a client or to help approve candidates. There are various other applications of the detection and/or quantitation of nucleic acids and new applications are being developed constantly.
The most common techniques to detect and measure nucleic acid analytes are based on the sequence-specific hybridization of the analyte with a complimentary nucleotide sequence probe which is marked with a detectable label, typically a fluorescent label, a radioactive label or another chemical label that can be detected in a secondary reaction. The probe is combined with the nucleic acid analyte, either in situ as part of a biological system or as isolated DNA or RNA fragments. The hybridization conditions are those that allow the probe to form a specific hybrid with the analyte, while not becoming bound to non-complementary nucleic acid molecules. The analyte can be either free in solution or immobilized on a solid substrate. The probe's detectable label provides a means for determining whether hybridization has occurred and thus, for detecting the nucleic acid analyte. The signal that is generated via the detectable sample can in some instances be measured quantitatively to back-calculate the quantity and the concentration of the analyte.
Current methods used to detect and measure nucleic acid analytes are briefly described below.
PCR Methods
The polymerase chain reaction (PCR) amplification of nucleic acids is regularly performed using fluorescently labeled oligonucleotide primers to produce an amplified DNA product that can be detected and quantified absolutely. A wide range of fluorochromes are now commercially available with spectral characteristics (λmax excitation and λmax emission) covering the wavelength range 350 to 700 nm, and into the near infra-red region of the electromagnetic spectrum. Thus, simultaneous, multiple detection of labeled molecules can be performed on the same sample, for example, following ‘multiplex’ PCR amplification of several nucleic acid sequences using pairs of oligonucleotide primers labeled with different fluorophores. Each pair gives rise to a separate amplified product that can be unambiguously identified due to its fluorescent label.
FISH Methods
Fluorescent in situ hybridization (FISH) is an important tool for clinical diagnosis and gene mapping. Labeled nucleic acid probes are used with multiple, simultaneous fluorescent detection to locate specific nucleic acid sequences in cells and tissues, and with the number of fluorochromes available there is the potential to visualize several fluorescent signals relating to different genetic sequences within the same sample.
Nucleic Acid Microarrays
Microarrays of nucleic acids that are prepared by combinatorial chemistry methods provide a convenient means to assay multiple, up to tens of thousands, analytes simultaneously. Typically, microarrays are probed with fluorescently labeled nucleic acid species, for example, from a clinical sample, and the position of any hybridized, labeled nucleic acid identified using fluorescence microscopy. The position relates to a known nucleic acid sequence immobilized at that part of the microarray.
Fluorescence Energy-Transfer (FRET) Methods
FRET relies upon the interaction of a fluorescent donor dye and a fluorescent acceptor dye, both of which are attached to the same molecule. If the donor and acceptor dyes are in proximity to one another, the acceptor dye quenches the fluorescent signal of the donor dye upon excitation. However, when the two dyes are held apart from one another, the fluorescence of the donor dye can be detected.
Molecular Beacon Methods
Molecular beacons are nucleic acid probes that contain both a covalently attached fluorescent dye and a non-fluorescent quencher moiety. Molecular beacons allow the diagnostic detection of specific nucleic acid sequences through their structural characteristics. The probes possess hairpin-forming regions, and in the absence of a complementary nucleic acid strand, the fluorescent dye and the quencher are in close proximity to one another and quenching of the fluorescent signal results. When incubated with a target nucleic acid analyte that possesses a complementary sequence, the probe anneals to the target, such that the fluorescent dye and the probe are held apart from one another, and fluorescence can be detected signifying the presence of a particular nucleic acid sequence.
Preferably, methods to detect and/or quantify nucleic acid analytes are carried out as homogeneous assays, which require no separate analyte manipulation or post-assay processing. Classically, agarose gel electrophoresis, possibly followed by Southern-blot hybridization or enzyme-linked immunoassays was used to detect and quantitate nucleic acid. Maniatis et al. (1982) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, NY, which is incorporated herein by reference in its entirety. Such procedures, and other methods that similarly rely on end-point analysis are generally labor intensive, require several separate and distinct handling processes and skilled personnel, are relatively slow to produce a result, and are prone to contamination and false positives due to the open system. In comparison, the advantages of a homogeneous assay, which represents a fully enclosed homogenous real-time detection system, include a faster turn-around time, especially when using microvolumes, higher throughput, better options for automation and multi-parallel analysis, and the use of a fully enclosed test system, which reduces the likelihood of contamination.
Homogeneous assays are particularly desirable with PCR methods and other amplification methods, because the amplification and the detection of the nucleic acid analyte can be carried out in one step without any risk of cross-contamination, which is a severe problem with all methods that rely on extensive amplification, especially in high-throughput analysis labs.
Prior art homogeneous detection and quantification methods for nucleic acid analytes involve a variety of chemistries and formats, which are exemplified below. Each of these methods is associated with certain disadvantages that create a need for improved detection/quantification strategies.
Hydrolysis Probes
Hydrolysis probes are described in Holland and Gelfand (1991) Proc. Natl. Acad. Sci. USA 88:7376-80 and U.S. Pat. No. 5,210,015, each of which is incorporated herein by reference in its entirety. This method takes advantage of the 5′-exonuclease activity present in the thermostable Taq DNA polymerase enzyme used in PCR (TAQMAN™ probe technology, Perkin-Elmer Applied Biosystems, Foster City, Calif., USA) and is amplified to homogeneous detection in PCR, as described by Heid et al. (1996) Genome Methods 6:986-94, which is incorporated herein by reference in its entirety. This method involves the use of a nucleic acid probe is used which is labeled with a fluorescent detector dye and an acceptor dye. Typically, the 2 dyes are attached to the 5′- and 3′-termini of the probe and when the probe is intact, the fluorescence of the detector dye is quenched by fluorescence resonance energy transfer (FRET). The probe anneals downstream of the amplification target site on the template DNA in PCR reactions. Amplification is directed by standard primers upstream of the probe, using the polymerase activity of the Taq enzyme. FRET quenching continues until the Taq polymerase reaches the region where the labeled probe is annealed. Taq polymerase recognizes the probe-template hybrid as a substrate, hydrolyzing the 5′ detector dye during primer-directed DNA amplification. The hydrolysis reaction releases the quenching effect of the quencher dye on the reporter dye, thus resulting in increasing detector fluorescence with each successive PCR cycle.
Mixed RNA/DNA sequence probes can also serve as hydrolysis probes to monitor PCR reactions, as described by Winger et al., U.S. Pat. No. 6,251,600 B1, which is hereby incorporated herein by reference in its entirety. The mixed RNA/DNA probes contain blocks of DNA nucleotides at either end of the probe and a stretch of RNA nucleotide sequence between the flanking DNA blocks. This type of probe also contains a detector and an acceptor dye, which are attached to the respective DNA blocks of the probe. Upon hybridization to a nucleic acid analyte, the resulting hybrid contains two stretches of DNA:DNA duplex structure, flanking a stretch of DNA:RNA duplex structure. In the presence of the enzyme RNAse H, the DNA:RNA duplex structure is subject to cleavage, because RNAse H specifically recognizes DNA:RNA duplexes and cleaves the RNA portion of these duplexes. As a result the two blocks of DNA nucleotide sequence of the probe are separated, which in turn results in an increased fluorescence of the detector dye, which is no longer quenched by the acceptor.
The efficiency of hydrolysis probes in homogeneous assays is generally limited by their inherent fluorescence background, which is caused by incomplete quenching. Fluorescence quenching in these probes is caused by fluorescence energy transfer (FRET), which decreases with the inverse sixth power of the distance between the donor and the acceptor. Since the two dyes of the FRET pair are not in close molecular proximity, the quenching in hydrolysis probes is inherently incomplete resulting in an observable fluorescence background and therefore in a low signal to noise ratio. Additionally, the efficiency of hydrolysis probes is highly dependent on the purity of the probes, because contamination with singly labeled probes results in unquenched fluorescence and therefore a high background.
Hairpin Probes
Hairpin probes or molecular beacons are described by Tyagi et at. (1996) Nat. Biotechnol. 14:303-308, and are applied to homogeneous detection in PCR, as described by Marras et al. (1999) Genetic Analysis 14:151-156, each of which is incorporated herein by reference in its entirety. Molecular beacons are nucleic acid probes that are able to form a hairpin substructure due to the presence of two inverted repeat sequences. They carry covalently attached detector and quencher dyes at the end of both arms of the hairpin substructure of the probe. This design allows for self-complementary hybridization of the two inverted repeat sequences to form a stable, hairpin structure in the absence of a specific target. The detector and quencher dyes are in close proximity to one another in this conformation, which results in quenching of the detector fluorescence. The stretch of nucleotide sequence between the inverted repeat sequences of a molecular beacon is complementary to its target, thus directing specific probe-target hybridization, which results in efficient separation of the quencher dye from the detector dye with subsequent light emission. Thus, in the presence of a complementary target sequence, the hairpin structure is eliminated and the separated dye fluoresces. No overlap is required between the emission spectrum of the fluorophore and the absorption spectrum of the quencher. This allows for a wider range of fluorophores to be used in molecular beacons as compared with hydrolysis probes (TAQMAN™ probes). Hairpin probes are most commonly used as “free-floating” probes to detect amplicons as they are produced by standard PCR amplification, but can also be attached to one of the primers to act as a self-probing beacon as described by Whitcombe et al. (1999) Nat. Biotechnol. 17:804-807, which is incorporated herein by reference in its entirety.
Hairpin probes are particularly difficult to design because their successful application requires several design conditions to be fulfilled simultaneously. Firstly, the two inverted repeats of the hairpin structure must have complementary counterparts in the target nucleic acid, which in turn requires the presence of inverted repeats in the target as well, a condition that is not generally met. Secondly, the Tm of the loop portion of the hairpin structure with a complementary nucleic acid sequence and the Tm of the stem portion need to be carefully balanced with respect to the temperature of the assay to allow the specific unfolding of the hairpin probe in the presence of the target without unspecific unfolding. Improper design of hairpin probes results in high fluorescence background and therefore a low signal to noise ratio. The efficiency of hairpin probes is particularly sensitive to the purity of the probes, because even minimal amounts of singly labeled impurities result in a high background in the assay.
Hybridization Probes
Hybridization probe design entails the use of two sequence-specific nucleic acid probes, each labeled with a fluorescent dye, one dye being a donor dye, the other dye being an acceptor dye. Typically, the two probes are designed to hybridize to a nucleic acid analyte close to each other in a head-to-tail arrangement that brings the two dyes into close proximity. In this arrangement, as demonstrated by Cardullo et al. (1988) Proc. Natl. Acad. Sci. USA 85:8790-04, which is incorporated herein by reference in its entirety, the fluorescence of the acceptor dye is enhanced if the donor is excited due to the radiationless uptake of energy from the donor. This method is applicable to PCR reactions (LIGHTCYCLER™ PCR technology, Roche Diagnostics, Indianapolis, Ind., USA), as demonstrated by e.g. Espy et al. (2000) J. Clin. Microbiol. 38:795-799, which is incorporated herein by reference in its entirety. For use with the LIGHTCYCLER™ instrument of Roche Diagnostics the 3′-end of one probe is labeled with fluorescein as a donor and the 5′-end of the other probe can be labeled with LC Red 640 or LC Red 705 as an acceptor. Upon the occurrence of FRET between the donor and the acceptor, the fluorescence of the acceptor is detected. The transfer of fluorescent resonance energy only occurs when both probes hybridize to the target in very close proximity, the optimal distance being one to five intervening bases between probes. Hybridization probes are used in conjunction with standard primers to direct the PCR amplification.
Assays based on hybridization probes require the design of two oligonucleotide probes and their synthesis and purification, which adds cost and increases the complexity of assays. The use of two different probes in each analysis is particularly disadvantageous in high-throughput settings where a multitude of samples need to be analyzed due to the linear relationship of the number of involved probes and the number of analyses to be performed. Additionally, assays based on hybridization probes are more difficult to multiplex due to the presence of a higher number of probes, each of which could potentially generate artifacts, such as false positives in a multiplexed analysis.
Probeless Detection
Probeless detection of nucleic acids takes advantage of the affinity of certain dyes for double stranded DNA. Ideally, a dye that is suitable for a probeless detection displays low or no fluorescence at all when not bound to double stranded DNA, but a bright fluorescence when attached to the DNA. Thus, upon binding of the dye to DNA, a fluorescent signal is generated that indicates the presence of the DNA. The binding of the dye occurs in a non-covalent manner and is not specific for the sequences of the DNA analyte. The method is applicable to PCR reactions where the presence or absence of amplicons can be monitored as the PCR reactions progress. Examples of probeless detection strategies for PCR reactions are exemplified by Higuchi et al. (1992) Biotechnology 10:412-417 and Wittwer et al. (1997) BioTechniques 22:130-138, each which is incorporated herein by reference in its entirety. Probeless detection strategies also involve the use of covalently linked dye pairs, wherein one of the dyes is a fluorescent intercalator, as described by Makino et al., U.S. patent application Ser. No. 2001/0014452A1. In this technique, the fluorescence of the intercalator is quenched by the second dye, the efficiency of the quenching being dependent on the presence of double stranded DNA. Upon the interaction of the covalently linked dye pair with double stranded DNA the quenching becomes less efficient and a fluorescence signal can be detected.
Probeless detection and quantitation strategies are inherently disadvantageous due to their non-specific nature. In general, these methods detect any kind of double stranded DNA regardless of the presence or absence of specific sequences. Therefore, probeless detection methods are prone to generate “false positives,” caused by e.g. the formation of primer dimers or non-specific amplification products in PCR reactions.
The detection of nucleic acid targets has also been described with a variety of other strategies that involve fluorescent detection. For example, Cardullo et al. (1998) Proc. Natl. Acad. Sci. USA 85:8790-04, describe the use of competitive hybridization probes, i.e. a pair of complementary oligodeoxynucleotides, each member of the pair being labeled with a covalently attached fluorescent dye at the 5′-terminus, which form a short stretch of double stranded DNA in the assay. The two dyes of the oligo-deoxynucleotide pair form a FRET system in which the fluorescence of the donor dye is quenched while the oligodeoxynucleotides are hybridized to each other. In the presence of a target nucleic acid analyte, the probes competitively hybridize with the target, which separates the two components of the FRET system resulting in observable fluorescence of the donor component. This method suffers from the disadvantage of being dependent on a FRET mechanism with the associated high fluorescence background. In addition, two probes are required per assay, which increases the complexity and the cost of the assay.
None of the described fluorescence based methods combines the desired features of homogeneous methods to detect and/or quantify nucleic acid analytes, i.e. high specificity, low fluorescence background and therefore a high signal to noise ratio, ease of probe design without restrictions caused by the sequence of the target, and low complexity associated with low cost.
The instant invention includes novel fluorescence based methods to detect and/or quantify nucleic acid analytes and novel nucleic acid probes that combine the desired features of homogeneous assays. The methods and probes of this invention have significant advantages and do not suffer from the limitations inherent to the prior art methods and probes. The nucleic acid probes described in this invention carry a multitude of covalently attached dyes in close molecular proximity and therefore have a very low intrinsic fluorescence background. They are highly sequence specific and not limited by complex design criteria, as for example hairpin probes, and are applied as a single probe per assay. They can easily be adopted in homogeneous assays, in particular in PCR based assays, and provide the results of the assays in real time. They are amendable to multiplexing in such assays and can be used as primers of a PCR reaction, which further simplifies PCR based assays. The probes are also applicable in assays conducted on nucleic acid microarrays. Furthermore, in one embodiment of the invention the probes provide switchable labels that can be activated and deactivated by an adjustment of the pH of the assay.
Probes that carry two covalently attached dyes in close molecular proximity have been described by Shinozuka et al. (1994) J. Chem. Soc. Chem. Comm. 1377-1378, which is incorporated herein by reference in its entirety. However, the probes disclosed by Shinozuka display a high fluorescence that is reduced upon the interaction with a complementary nucleic acid target. These probes, despite their usefulness in general studies of nucleic acid association and hybridization, cannot be applied effectively in homogeneous assays because of their intrinsic high fluorescence. The probes of this invention have a very low intrinsic fluorescence and are therefore superior to the prior art probes.